The miR-183/96/182 Cluster Regulates Macrophage Functions in Response to <b><i>Pseudomonas aeruginosa</i></b>

Abstract

Macrophages (Mϕ) are an important component of the innate immune system; they play critical roles in the first line of defense to pathogen invasion and modulate adaptive immunity. MicroRNAs (miRNAs) are a newly recognized, important level of gene expression regulation. However, their roles in the regulation of Mϕ and the immune system are still not fully understood. In this report, we provide evidence that the conserved miR-183/96/182 cluster (miR-183/96/182) modulates Mϕ function in their production of reactive nitrogen (RNS) and oxygen species (ROS) and their inflammatory response to Pseudomonas aeruginosa (PA) infection and/or lipopolysaccharide (LPS) treatment. We show that knockdown of miR-183/96/182 results in decreased production of multiple proinflammatory cytokines in response to PA or LPS treatment in Mϕ-like Raw264.7 cells. Consistently, peritoneal Mϕ from miR-183/96/182-knockout versus wild-type mice are less responsive to PA or LPS, although their basal levels of proinflammatory cytokines are increased. In addition, overexpression of miR-183/96/182 results in decreased production of nitrite and ROS in Raw264.7 cells. We also provide evidence that DAP12 and Nox2 are downstream target genes of miR-183/96/182. These data suggest that miR-183/96/182 imposes global regulation on various aspects of Mϕ function through different downstream target genes.

MicroRNA-96 Promotes Vascular Repair in Oxygen-Induced Retinopathy—A Novel Uncovered Vasoprotective Function

“…For instance, miR-96 in human retinal pigment epithelial cell stimulated with hydrogen peroxide (ROS inducer), was found to be strongly downregulated (Ayaz and Dinc, 2018). Conversely, overexpression of the miR-183/96/182 cluster resulted in a decreased production of nitrite and ROS by macrophage (Muraleedharan et al, 2019) and a neuroprotective role in the brain, by positively regulating glutathione (GSH) levels, a major tissue antioxidant (Kinoshita et al, 2014). Collectively, these studies support the possibility that miR-96 can be decreased in hyperoxic/ oxidative stress conditions; this does not exclude other mechanisms such as extracellular vesicles (Liu and Lu, 2015;Battaglia et al, 2019).…”

MicroRNAs in Ocular Infection

“…One of the most prominent findings is that miRNAs modulate the ocular infection through its regulation on innate and adaptive immunity. In PA keratitis, both miR-155 [132] and the miR-183/96/182 cluster [136,137] have been shown to suppress phagocytosis and intracellular bacterial killing of PA by neutrophils and Mϕ. Knockout or knock-down of the function of miR-155 and the miR-183/96/182 cluster resulted in increased phagocytosis and bacterial killing, which contributes to the decreased severity of PA keratitis in the ko mice.…”

“…For miR-155, Yang et al showed that Rheb, which can directly interact with mTOR and increase mTOR activity [133,134], is targeted and mediates the regulation of miR-155 on bactericidal capacity of innate immune cells [135]. In contrast, for the miR-183/96/182 cluster, Nox2, one of the key enzymes required to generate superoxide and other bactericidal ROS and RNS, is targeted by miR-182 and 96, contributing to its effect on phagocytosis and bacterial killing by Mϕ and neutrophils [136,137].…”

“…Recently, with both in vivo and in vitro approaches, we showed that the conserved, paralogous miRNA cluster, the miR-183/96/182 cluster (miR-183/96/182), modulates the corneal response to PA infection through its regulation of pathogenesis of the disease at multiple levels [136,137,138,139]. Initially, we and others identified miR-183/96/182 as a sensory organ-specific miRNA cluster, as it is highly specifically expressed in all major sensory organs [140,141,142] and is required for normal development and functions of all major sensory domains [53,140,143,144,145,146,147].…”

“…Inactivation or knockdown of miR-183/96/182 resulted in increased phagocytosis and intracellular bacterial killing capacity of both Mϕ and neutrophils [136]. This effect could be the result of increased production of reactive nitrogen species (RNS) and reactive oxygen species (ROS) in these innate immune cells through its regulation of Nox2, a key enzyme required for the generation of superoxide (O 2 − ) and other microbicidal ROS and RNS, as seen in Table 1 [137,148,149]. In addition, we showed that miR-183/96/182 promotes the production of pro-inflammatory cytokines in Mϕ through targeting DAP12 [137], which is consistent with the observation of an overall decreased level of pro-inflammatory cytokines in the cornea in response to PA infection [136].…”